Polymeric Gene Delivery to Cord Blood Derived Mesenchymal Stem Cells (CB-MSC)

Kucharski, Cezary (University of Alberta)
Rose, Laura (University of Alberta)
Uludag, Hasan (University of Alberta)


Polymeric delivery vehicles are investigated for gene delivery due to their better safety profile over viral systems, which can induce excessive inflammation and immune responses, as well as oncogenicity in certain situations. The cationic charge of the polymers interacts with the anionic DNA and forms nanoparticles suitable for gene delivery and cellular uptake. A wide range of cationic polymers has been explored for gene delivery over the years. We found that small (2 kDa) polyethylenimine (PEI) modified with linoleic acid (LA; PEI-LA) are effective gene delivery agents in vitro in a variety of cells [1,2,3]. These polymers are relatively small in size (so that their toxicities are minimal), but are effective in gene delivery due to incorporation of lipid groups in the polymer. The lipid groups results in lipophilic nanoparticles that allow relatively efficient transport into cells through the cell membrane. Other non-viral vectors are commercially available; however, they are often toxic to cells they aim to transfect. In this study, we explored the utility of PEI-LA for modification of cord-blood derived mesenchymal stem cells (CB-MSCs), an important cell phenotype for tissue engineering purposes. We sought to assess transfection of Green Fluorescent Protein (GFP) by using lipopolymers as a reporter gene, as well as to explore the expression of Bone Morphogenetic Protein-2 (BMP-2) as a therapeutically relevant gene.

Materials and Methods

The preparation of PEI-LA was described previously [2]. For GFP expression, complexes were prepared by mixing gWiz-GFP plasmid (Aldevron) with PEI-LA at a weight ratio of 2.5:1 (PEI-LA to plasmid, w/w). Complexes of blank gWiz plasmid and PEI-LA were set as negative control. As a commercial carrier, Lipofectamine 2000® were used for comparison purposes since this liposomal carrier was found to work generally well with a wide range of cells in our hands. Complexes were tested in the primary human CB-MSC in MEM Alpha (Gibco®) with 10% serum and 1% penicillin/streptomycin. Cells (passage 6-8) were exposed to gWiz or gWiz-GFP complexes for 4 hours followed by replacement of the medium containing complexes with fresh medium. Cells were trypsinized on day 3 for analysis with flow cytometry, where untreated cells were used to represent 1% autofluorescence. Similarly, transfections were conducted with a BMP vector (gWIZ-BMP2) to determine transfection of a therapeutically relevant gene [1]. BMP-2 protein concentrations in supernatants were determined after 3 days of transfection using a commercially available ELISA kit (R%26D Systems).


Plasmid transfection in the PEI-LA/gWiz-GFP group resulted in a 5-fold increase in the mean GFP fluorescence, compared to control (PEI-LA/gWiz) group. Transfection in the Lipofectamine/gWiz-GFP group was even more effective with 15-fold increase in the mean fluorescence compared to control (Lipofectamine/gWiz) group (Figure 1A). Also, the percentage of modified cells was 12% higher in PEI-LA/gWiz-GFP group, and 21% higher in Lipofectamine group compared to the Control group (Figure 1B). These results indicated ready transfection of the CB-MSC but the commercially available liposomal carrier appeared to be superior to the polymeric carrier in these cells. Transfection affected <30% of the cells, which is adequate for hard-to-transfect primary cells but could be improved for a more efficient therapeutic approach. We next investigated transfection of a BMP-2 expression plasmid in CB-MSC (Figure 2). Unlike the GFP expression, there was very little, if any, secretion of BMP-2 into the medium whether PEI-LA or Lipofectamine 2000 were used for transfections. To ensure that the designed plasmid was functional, transfections were carried out in 293T kidney fibroblast cell line; in this case, a robust amount of BMP-2 secretion was readily seen with both carriers, indicating that the designed plasmid was functional for BMP-2 secretion.

Discussion and Conclusion

Although GFP expression in CB-MSC is encouraging, the low level of BMP-2 secretion is concerning for the feasibility of using these cells for gene therapy purposes. It will be important to determine the influence of cell passage on transfection efficiencies, since the cells derived for this study was from a later passage (6-8). Measures to enhance transfection efficiency by using superior formulations of polymeric carriers might need to be also formulated in order to achieve better transfection efficiencies. Future work in this direction is currently under-investigation.


We thank the CIHR, NSERC and AIHS for financial support of this project. We also thank J. Fife for synthesizing the polymers and Dr. LA. Marquez-Curtis and Dr. Janowska-Wieczorek for providing the CB-MSC for this study.


[1] Wang et al., J Surg Res. 2013 183(1):8-17. [2] Neamnark et al., Mol Pharm. 2009, 6:1798-815. [3] Rose et al., Biomaterials. 2012, 33:3363-74.

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